As described in project 1, the highly conserved Wnt/beta-catenin signaling pathway is a crucial pathway for embryogenesis. Mutations in oncogenes or tumor suppressors in the Wnt/beta-catenin pathway lead to its constitutive activation and cause cancer in the adult. Alternative Wnt signaling pathways, controlled by noncanonical Wnts such as Wnt5a, have been shown in vitro to antagonize Wnt/beta-catenin signaling suggesting that stimulation of these alternative pathways could function to suppress tumorigenesis. We have previously shown that embryos lacking Wnt5a display defects in the elongation of the PS and PSM (as well as the limb, face, brain and gut) that are distinct from Wnt3a-/- phenotypes. Proper elongation of the PSM is important for correctly establishing the slope of the Wnt3a gradient that controls mesodermal stem cell homeostasis and somitogenesis. Although several pathways have been proposed to transduce Wnt5a signals, our genetic and reporter analyses demonstrate that Wnt5a interacts with core components of the Planar Cell Polarity (PCP) pathway (Vangl2/Looptail(Lp) and Dvl2) to regulate PSM elongation. Many in vitro studies have suggested that Wnt5a negatively regulates the Wnt/&amp;#946;catenin pathway however analyses of Wnt/&amp;#946;catenin target genes and reporters in Wnt5a-/- embryos were not supportive. To address the mechanisms of Wnt5a signaling, we transcriptionally profiled Wnt5a mutants following the same procedure that we successfully used to profile Wnt3a mutants. In contrast to our results with Wnt3a mutants, no consistent changes in gene expression were observed when comparing wt and Wnt5a-/- profiles suggesting that Wnt5a does not regulate transcription and does not inhibit the expression of Wnt3a/&amp;#946;catenin target genes. Together, our data support the interpretation that Wnt3a and Wnt5a regulate distinct pathways in vivo, and that Wnt5a signals through the poorly understood mammalian PCP pathway to regulate PSM elongation. Null alleles of Wnt5a result in numerous phenotypes and neonatal lethality. To better investigate the function of Wnt5a in specific embryonic and adult tissues we have generated conditional loss and gain-of-function alleles of Wnt5a. Early results indicate that the conditional LOF allele is working as expected and is already yielding interesting results. We have previously shown in null mutants that Wnt5a is required for proper development of the face, kidney, and heart (manuscript in preparation), and have recently published a paper describing the role of Wnt5a in gut elongation (Cervantes et al., 2009). Interestingly, the kidney, gut and heart phenotypes are recapitulated in T-Cre;Wnt5aflox/&amp;#916;mice while the face phenotype is rescued. Although preliminary, these results suggest that the heart phenotype, for example, arises strictly from a requirement for Wnt5a in the outflow tract. The kidney phenotypes are currently being analysed in collaboration with my colleague and kidney expert, A. Perantoni. In an effort to better understand the mechanisms of Wnt5a/PCP signaling, we are currently attempting to develop an in vitro morphogenesis assay by generating ES cells carrying epitope-tagged inducible Wnt5a, Vangl2, Ror2 (an atypical Wnt5a receptor), Prickle, and mutated alleles thereof. Several ES cell lines have been generated and are currently being characterized. Differentiating embryoid bodies generate self-organizing asymmetry and display polarized distribution of differentiated cell types. We will assess asymmetric gene expression, differentiated cell distribution, adhesion and embryoid body morphology upon overexpression of Wnt5a &amp;PCP genes in embryoid bodies. Epitope tags will be used to IP specific PCP proteins from embryoid bodies and associated proteins will be determined by mass spec. We are also addressing the pathways that function upstream of Wnt5a. Our transcriptional profiles of Wnt3a mutants showed that several components of the PCP pathway were down-regulated, suggesting that Wnt3a/&amp;#946;catenin signaling may, directly or indirectly, regulate non-canonical Wnt signaling. Remarkably, during the characterization of our Msgn-inducible ES cells, we found that Wnt5a is rapidly, and dramatically, activated upon Msgn1 induction, suggesting that Wnt5a may be a target gene of Msgn. We are currently examining Wnt5a expression in Msgn-/- mutants, and characterizing the Wnt5a promoter, focusing on Ebox sites that could be directly regulated by Msgn. Together with the identification of Msgn target genes by the integrative genomics experiments described in project 1, we expect to gain a better understanding of the interactions between the Wnt/&amp;#946;catenin and PCP pathways. We have also identified mouse homologues of Daam (Dishevelled (Dsh)-associated activator of morphogenesis), a Formin protein that promotes actin polymerization and which interacts directly with Dsh and Rho to control convergent extension in frogs. We have generated numerous tools including targeted conditional loss-of-function alleles, LacZ-tagged alleles, tagged cDNAs and antibodies to understand how the Daam proteins function in the Wnt pathway to regulate cell and tissue polarity during mouse embryogenesis. We have found that our Daam1 loss of function mutants result in embryonic lethality at mid-gestation due to defects in the development of the placenta. We are continuing to characterize the mutant placentae and expect to write a manuscript in the coming months